Long-term preservation strategy for legume root nodule phenotypes coupled with a comprehensive evaluation method.

Abstract

Nodule color and morphology are key readouts of legume symbiotic performance. However, long-term preservation of post-excavation nodules with intact morphology, color, and microbial cleanliness remains a major challenge. This study developed a two-stage aqueous-phase preservation method (TAPP) that enables rapid structural fixation and long-term chemical stabilization. A comprehensive evaluation was subsequently established, incorporating composite morphological score (0-5), color difference (ΔE) and its piecewise slope over time ([Formula: see text]), and visible contamination grade (0-3). Peanut and soybean nodules from multiple regions and cultivars were tracked for 24 months under five preservation methods: TAPP, FormalinCu, TAPP-Resin, Resin, and AirDry. TAPP showed the best overall preservation, with composite morphological scores of 4.65 ± 0.14 for peanut and 4.63 ± 0.22 for soybean at 24 months, and no visible mold. Color change slowed over time: [Formula: see text] decreased from 1.83 to 1.10 ΔE·month - 1 during 0-1 month to 0.16 ΔE·month - 1 during 12-24 months, yielding final ΔE values of 10.53 ± 1.88 and 10.32 ± 1.93, respectively. Notably, TAPP pretreatment markedly improved resin-embedded samples, demonstrating scalability and flexible deployment. In addition, this study further proposes a stage-wise workflow that integrates on-site pre-fixation, long-distance transport, and long-term storage to enable cross-regional circulation and collaborative phenomics of oxidation-prone, dehydration-sensitive nodules. Together, this work establishes a standardized, traceable workflow to preserve and benchmark legume root nodule phenotypes, supporting cross-laboratory comparability and longitudinal cross-source analyses.

Plant phenotyping relevance

マメ科根粒の形態・色・汚染状態という植物表現型を長期保存し、定量評価・比較する手法と標準化ワークフローが研究の中心であるため。

Code and data availability

The paper's nodule phenotype datasets (morphology scores, ΔE color measurements, contamination grades over 24 months) are not publicly deposited; the Data availability statement says they are available only from the corresponding author on reasonable request. No author analysis code, scripts, models, or public data URL

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